Fluorophores Under Glass

What is 30 times brighter than fluorescent markers, impervious to photobleaching, and won't leach heavy metals and other toxins into biological samples?

Bennett Daviss
Sep 11, 2005
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© Cornell University

Schematic representation of a Cornell Dot, with several molecules of a fluorescent rhodamine dye encapsulated in the center. The dye has been modified with a group that links to the encapsulating silicon.

What is 30 times brighter than fluorescent markers, impervious to photobleaching, and won't leach heavy metals and other toxins into biological samples? Why, Cornell dots, of course – Ulrich Wiesner's attempt to make a cheaper, safer, and brighter biological marker.1

Cornell dots, named for the university where Wiesner is a professor of materials science and engineering, are molecules of fluorescent dyes encapsulated in a silica shell. Wrapping the dye in glass keeps the dyes' fluorophores from dissolving in the biological sample. The surface of the glass beads can be coated with any of a variety of ready-made "targeting molecules" (for instance, antibodies) matched to specific sites in a sample. By aggregating the beads at those sites of interest, the glass spheres can create a signal as much as 20 times brighter than ordinary dyes.

The brightness also results from grouping several dye molecules in one silica capsule. Adjacent dye molecules can dim each others' lights. But Cornell dots' fluorophores are separated by, and firmly attached to, the silica casing, allowing each molecule to shine to its fullest. The casing shields the fluorophores from water, where excited fluorescent molecules can generate singlet oxygen, a highly reactive radical that can lead to photobleaching.

Wiesner's dots are also cheap and easy to make, requiring only a one-step, room-temperature process using water-based or alcohol solvents. "I think it is a breakthrough," says Stephane Petoud, a chemist who works with luminescent lanthanides at the University of Pittsburgh.

Still, Petoud and others express reservations about Cornell dots' inherent girth, which ranges from 20 to 30 nm. "These Cornell dots certainly will be more stable than conventional dyes," says Simon Watkins, director of the University of Pittsburgh's Center for Biologic Imaging, but they are large; more than twice the size of a typical dye molecule. "The efficiency of the labeling process decreases as the size of the tag increases. You can't necessarily be sure you're attaching the tag to the right thing," adds Watkins. It would be tricky, for instance, to firmly mate a 25-nm tag to a protein less than 10 nm in size.

"If you had an antibody attached to a label of that size and were trying to get the antibody to bind to something inside a chromosome or complex cell, that big label might keep the antibody from penetrating into those areas to reach the target," says Alan Waggoner, director of the Centre for Light Microscope Imaging and Biotechnology at Carnegie Mellon University.

<p>FLUORESCENT CORE-SHELL SILICA NANOPARTICLES:</p>

© 2005 American Chemical Society

(A) 30-nm core-shell fluorescent silica nanoparticles Cornell Dots) as viewed under transmission electron microscopy. (B) The synthesis protocol can be extended to incorporate organic dyes with different spectral characteristics, covering the entire UV-vis absorption and emission wavelengths. Organic dyes incorporated from left to right are Alexa 350, N-(7-(dimethylamino)-4-methylcoumarin-3-yl), Alexa 488, fluorescein isothiocyanate, tetramethyl-rhodamine isothiocyanate, Alexa 555, Alexa 568, Texas Red, Alexa 680, and Alexa 750. (From H. Ow et al., Nano Letters, 5:113–7, 2005.).

Wiesner argues his dots are no bigger than quantum dots and notes his group has already demonstrated the tags' practicality. The researchers attached an antibody to the dots' surface that connected with rat basophilic leukemia mast cells. "We put these antibodies on the surface of Cornell dots and showed that we could specifically label those cell surface receptors on mast cells and then take pictures of them," Wiesner says. "This is the first step in using our dots for bioimaging."

But it isn't the last. By infusing a single sample with Cornell dots topped with a variety of surface-targeting molecules, Wiesner plans to make movies of biological processes in situ. "You not only want to visualize things on a molecular level, but you also want tags to function as reporters," he states. For example, a drugmaker testing a new candidate would like to see where in a cell, and in what sequence, the candidate triggers certain biochemical events. He hopes to complete a paper on the dots' use as reporters within a year.

Cornell physicist Watt Webb, who collaborated with Wiesner to create and evaluate the dots, says quantum dots, which, unlike Cornell dots, blink on and off, can take perhaps four times as long as Cornell's version to fluoresce after stimulation. But Charles Hotz, vice president of research and development for the Quantum Dot Corp. in Hayward, Calif., isn't looking over his shoulder. He notes Wiesner's group still hasn't published data about Cornell dots' photostability under harsh conditions and points out that, because the dots are still dye-based, users can't excite a spectrum of colored signals with a single light source, as they can with quantum dots.

"A Cornell dot is still a dye molecule," Hotz says, "but it improves on dye molecules' properties in good and useful ways."